Methods for biochemically phosphorylating nucleoside to produce nucleoside-5'-phosphate ester by using the following phosphate group donors are known, including a method which uses p-nitrophenyphosphoric acid (Japanese Patent Publication No. 39-29858), a method which uses inorganic phosphoric acid (Japanese Patent Publication No. 42-1186), a method which uses polyphosphoric acid (Japanese Patent Laid-open No. 53-56390), a method which uses acetylphosphoric acid (Japanese Patent Laid-open No. 56-82098), and a method which uses adenosine triphosphate (ATP) (Japanese Patent Laid-open No. 63-230094). However, these methods have not been satisfactory to produce nucleoside-5'-phosphate ester efficiently and inexpensively because the substrates to be used are expensive, or because by-products are produced in the reaction.
Thus the present inventors have developed a method for efficiently producing nucleoside-5'-phosphate ester without by-producing 2'-, 3'-nucleotide isomers by allowing cells of a specified microorganism to act under an acidic condition on a nucleoside and a phosphate group donor selected from the group consisting of polyphosphoric acid or a salt thereof, phenylphosphoric acid or a salt thereof, and carbamyl phosphate or a salt thereof (Japanese Patent Laid-open No. 7-231793).
However, even this method has had the following drawbacks. Namely, for example, a part of the substrate is degraded during the reaction due to a nucleoside-degrading activity which unfortunately exists in a slight amount in the cells of the microorganism to be used. Moreover, if the reaction is continued, produced and accumulated nucleoside-5'-phosphate ester is degraded. Therefore, by-products are produced in a reaction solution, and it has been impossible to obtain a sufficient yield. In addition, the reaction cannot be performed if the substrate is added at a high concentration because of a low transphosphorylation activity per microbial cell.